Perivascular pathways

Routes of perivascular efflux
Some of the possible routes for perivascular movements of solutes are indicated in Fig. 5. Whether or not actual fluid filled spaces exist around the blood vessels, it is believed that substances can move along preferential routes parallel to the blood vessels. (The description that follows is primarily for grey matter. As suggested originally by Rosenberg et al. in 1980 [65] there are likely to be preferential routes for fluid movement parallel to axons in white matter. It should also be noted that there may be regional variations, see e.g. [66, 67]). The idea that the basement membranes of microvessels can provide a preferential route stems from observations that when horseradish peroxidase is introduced into CSF with consequential influx along arteries the peroxidase is found to be localized in the basement membranes around microvessels. The idea has subsequently been supported by similar observations for other macromolecules (see e.g. [16, 68–72]). However, calculations by Asgari et al. [73] imply that unless the matrix of the microvascular basement membranes has a resistance substantially less than a sleeve of ®Matrigel with the same dimensions, they will not provide a preferential route for fluid flow parallel to the microvessels. A preferential route for movement along the vessels does not conflict with the movements of solutes outward by diffusion into the surrounding interstitial fluid. Regardless of whether or not the microvessel basement membranes provide a route with relatively low resistance, the distance from anywhere in the parenchyma to the nearest larger vessel is still likely to be relatively small, e.g. 100–200 µm. (Striking images of the vascular tree can be seen in [72]). For distances this short, diffusion is expected to be the dominant mechanism of extracellular movement [16, 24, 72, 74–81].
Fig. 5 Diagram indicating putative perivascular routes for substances to move into, out of and through the brain parenchyma. The lumens of arteries, arterioles, venules and veins are surrounded by a layer of endothelial cells with a basement membrane, then a layer of vessel wall including smooth muscle, and outside that there may be a further perivascular space with fluid and connective tissue bounded by basement membranes of the smooth muscle, pial and glial cells. Close to the surfaces of the brain these further spaces are often called Virchow-Robin spaces. Movements parallel to the large vessels may be intramural, through the extracellular space of the vessel wall, or extramural either in the outermost basement membranes or, in the opinion of some workers, in a fluid filled space. In this review both intramural and extramural pathways are called perivascular routes. Parallel to microvessels movement may be preferentially within the basement membrane separating the endothelial cells from the glial endfeet or it may be more diffuse through the interstitial spaces between the parenchymal cells
Markers for perivascular transport clearly have perivascular pathways for entry and exit from the parenchyma, but there is controversy as to whether efflux, influx or both occur along arteries and/or veins (for discussion see [16, 39, 41, 52, 72]). Efflux along arteries has been seen in many studies (e.g. [70, 82–88]) with substances even reaching the large arteries near the circle of Willis [82], and influx has also been seen in many studies [15, 16, 25, 69, 71, 79, 84, 88–92]. Evidence of influx along some vessels was obtained as early as 1960 [93]. Perivenous influx [16] and efflux [25, 69, 84, 94] have been reported. Efflux along unspecified blood vessels has also been seen [79]. The available evidence suggests that both influx and efflux occur along both arteries and veins [41, 78, 95] either via common pathways or separately along parallel pathways [88, 95] (see Proposal 2 below). In Fig. 5 movements are shown as occurring in both directions along both.
There has also been disagreement over which of the structural components of the arteries provide the principal routes for periarterial transport with some favouring an extramural, fluid filled perivascular space, possibly containing connective tissue fibres [16], between the vessel walls and the astrocyte endfeet, see e.g. [25, 71, 78, 79, 81, 83–85, 87, 92, 96]5 while others favour the view that “perivascular spaces” are not fluid filled, free spaces but rather perivascular pathways via basement membranes either within the smooth muscle layer or on the outside surface of the artery [52, 70, 72, 88, 97–99] (see Fig. 5).
Free spaces may be highly compressible, allowing modest changes in pressure to change their dimensions as envisaged in the proposal that variations in the blood pressure within the vessels somehow drive perivascular movements. By contrast basement membranes are likely to be much less compressible and are likely to offer much greater resistance to flow (see [73, 100, 101]), thus precluding blood pressure variations as the driving force for perivascular flow (see next section). Diem et al. [100] have proposed vasomotion as an alternative. Pizzo et al. [16] have suggested that both basement membrane routes and other, extramural routes exist with their relative importance depending on the size of vessel and the size of the solute. Another proposed variation is a hybrid with an extramural basement membrane route mediating fluxes into the brain and an intramural basement membrane route between smooth muscle cells mediating fluxes outwards [88, 95].
It is quite evident that solutes even as large as amyloid-β have access to the basement membranes between the smooth muscle cells (see e.g. [16, 70, 93, 102]), but it is not known whether the solutes reach these locations via an intramural route with movement along basement membranes as favoured by Carare, Weller, Hawkes and colleagues [70, 88, 95] or via extramural pathways with subsequent penetration from these into the basement membranes within the vessel wall (see Figure 21 in Sect. 5.7.1.2) or some mixture of the two. Arbel-Ornath et al. [87] used two-photon imaging to investigate the position of a 3 kDa fluorescent dextran during efflux following injection into the parenchyma. Shortly after injection they saw fluorescence within the parenchyma, in perivascular spaces surrounding small arteries and, at lower concentration, between the smooth muscle cells.
There has been controversy about the nature of the connections between the perivascular spaces adjacent to larger blood vessels within the parenchyma, the CSF and the perivascular spaces of the vessels passing through the subarachnoid spaces [1, 16, 25, 54, 71, 72, 81, 103–109]. However, whatever the exact perivascular pathway used, solutes exiting from the parenchyma along perivascular routes appear to be effluxed partly to CSF in the basal cisterns or subarachnoid spaces and partly to the outer meninges [85] and/or lymphatics [94, 107, 109–115]. Movement of small solutes and water does take place between fluid in the subarachnoid space and fluid within the perivascular spaces (see Section 4.1.1.1 of [41]). However a substantial proportion of perivascular efflux of large solutes appears to pass to lymph without first appearing in CSF in the cisterna magna6 (see Fig. 6) [16, 39, 52, 82, 83, 94, 96, 105, 107, 111, 115–119].
Fig. 6 Schematic diagram indicating possible routes for efflux of large solutes from the parenchyma along perivascular routes. a Large solutes emerging from the parenchyma via intramural or extramural routes along arteries (and possibly veins) may either mix with CSF or continue along the walls of blood vessels. The blood vessels span the subarachnoid space (see Figs. 1 and 6) before leaving the brain to reach the rest of the body. The fluid that continues along these vessels may enter either blood or lymph, but solutes as large as serum albumin will enter only lymph. b Large solutes that have reached CSF will be taken to sites of CSF outflow including the arachnoid villi, where the solutes will enter venous blood, and the cribriform plate, where they will enter lymph. (Based primarily on data for radio-iodinated serum albumin RISA [82, 83, 125] and on the location of the pia surrounding arteries taken from [103]). The anatomical relations of the pathways or spaces remain controversial
Those solutes that do reach CSF from the parenchyma can be taken out of the cranium via CSF outflow. Routes for CSF outflow were reviewed comprehensively by Pollay in 2010 [119] This outflow is partly via arachnoid villi, partly via perineural routes including those across the cribriform plate to the nasal mucosa [119–121] and possibly also via extra-parenchymal perivascular routes (see Fig. 6) [16, 81, 105, 111, 119, 122–124]. Outflow via arachnoid villi leads directly to venous blood while outflow via the cribriform plate may deliver solutes directly to lymphatics or to the extracellular fluid in the nasal mucosa [118, 121, 125]. Small solutes (e.g. lactate) and solutes even as large as inulin may leave the nasal mucosa by entering blood across peripheral capillary walls but larger solutes (e.g. albumin) will leave via lymph flow to cervical lymph nodes [125]. Outflow via other routes leads at least in part to lymph (see e.g. [111]).

Mechanisms driving perivascular solute efflux
Diffusion is not adequate for perivascular influx because substances added to CSF are found deep in the parenchyma much too quickly for diffusion over the distance involved, a millimeter or more [25, 68, 69, 84]. Similarly diffusion cannot account for efflux from parenchyma to CSF of substances like polyethylene glycol and dextran [126, 127], serum albumin [83], mannitol [25] or inulin [62, 128]. Thus alternative mechanisms have been proposed (see Fig. 7).
Fig. 7 Proposals to explain rapid transfer of markers via periarterial spaces: a the original proposal; b proposed perivascular convection and interstitial diffusion c the glymphatic proposal. In a the blood–brain barrier secretes fluid which flows out of the parenchyma via preferred routes (here the perivascular routes). In b transport in the perivascular spaces is assisted by convective stirring or mixing. In c (see Figure 5 of Iliff et al. [25]) there is preferential inflow via the space between the arterial wall and the pial sheath and preferential outflow via spaces surrounding veins. Red lines represent pial membranes, grey lines the layer of glial end-feet or glia limitans, black arrows are fluxes of markers carried or assisted by convection, green arrows are primarily diffusion. The location of the pial barriers is based on Zhang et al. [23]. The anatomical basis of the perivascular spaces remains controversial
(Modified from Figure 9 in [41])
Proposal 1 The first proposal (Fig. 7a) was that secretion of fluid by the blood–brain barrier provides a small pressure gradient for outflow of ISF along preferential routes (see [83, 126, 127, 129, 130]). These routes could be perivascular spaces or the extracellular spaces parallel to the axons in nerve fibre tracts. When this proposal was put forward more than 30 years ago (see e.g. [83]) it was believed that the half-life for clearance of marker solutes by outflow was of the order of 12 h. However, all of these early studies were performed on animals anaesthetized using barbiturates. Using either conscious animals or those anaesthetized with ketamine/zylazine or halothane, the half-lives are much shorter, 2–4 h [25, 62, 85, 131]. Perivascular efflux of solutes is considerably faster than envisaged by Cserr and coworkers. It should also be pointed out that Proposal 1 does not and was never intended to provide any explanation for the rapid influx of solutes. In Proposal 1 (and in Proposal 3, see below) the solutes are swept out of the parenchyma by the flow through the perivascular system. Estimates of the flow rate required to eliminate substances at the observed rates can be calculated from their clearances1 CLperivascular=rateofelimination/cisfand the assumption that the concentration of the solute is the same in ISF and the outflow. Then because elimination is by outflow2 rateofelimination=rateofoutflow×concentration in outflowand substituting that into the definition of clearance,3 CLperivascular=rateofoutflow×concentration in outflow/cisf,which, because the concentration in the outflow is the same as cisf, becomes4 CLperivascular=rateofoutflow.
From the known volume of distribution of suitable substances such as inulin or sucrose, 200 µL g−1, and the range of their half lives, 2–4 h, and the relation between clearance, half-life and volume of distribution, CL = 0.69 VD/t1/2, the clearances and thus the required flow rates are in the range 0.6–1.2 µL g−1 min−1. For a human with a 1400 g brain this is 1.2–2.4 L day−1. Even the bottom of this range is somewhat more than twice the rate of production of CSF. There is no other reason to suspect that there is a rate of secretion of fluid across the blood–brain barrier that exceeds the rate of fluid secretion by the choroid plexuses (see Section 4.1 in [4]). The rate of fluid secretion across the blood–brain barrier is very unlikely to be this large and is almost certain to be insufficient to account for perivascular clearance of solutes.
Proposal 2 (Fig. 7b) The second suggestion, recently revived, is that convection in the perivascular spaces, arterial and possibly venous, leads to convective mixing of the fluid in the spaces allowing relatively rapid movements of solutes both inwards and outwards [41, 78, 82, 96, 132]. Such mixing probably presupposes that perivascular spaces are compressible. Convective mixing is perhaps better called dispersion [78]. Papisov [133] and Asgari et al. [134] discuss a similar effect in the spinal cord allowing transport of solutes down their concentration gradients against the direction of net flow of CSF and at rates much greater than allowed by diffusion. In this proposal diffusion is taken to be adequate to explain movements within the interstitial spaces in the parenchyma because the distances involved are sufficiently short (see Sect. 3.2.1).
In this proposal (and in Proposal 3, see below), an important part of the mechanism is thought to be convection in spaces whose dimensions are changed by periodic compression resulting from the changes in blood pressure during the cardiac cycle [13, 25, 70, 82, 96, 132]. The length of space around a cortical vessel that is compressed at one time is as long as the vessel [78, 82]. Bradbury et al. [82] were of the opinion that periodic compression and reexpansion of this space “would cause to-and-fro movement of fluid in and out of the brain” such that “A basis would be provided for substances in solution or suspension to be moved either out of or into the brain depending on the relative concentration in subarachnoid CSF.” Another variation on this theme may be possible if there are layers of differing compressibility, both connected via relatively low resistance pathways to the brain surface.
Back-and-forth convective movements in perivascular spaces would only be apparent using techniques with both good spatial resolution and time resolution better than a fraction of a second. Such movements have been observed in perivascular spaces very close to the cortical surface using india ink [84] and in the periarterial spaces at the cortical surfaces using microspheres [108]. But with techniques now available for viewing, if perivascular spaces exist that allow convective back and forth movements, all that would be seen within the parenchyma would be accelerated movement down the concentration gradient regardless of its direction, i.e. the periarterial influxes and effluxes that have been observed.
Proposal 3 (Fig. 7c) The third proposal, the glymphatic hypothesis [25, 109, 135–137], asserts.There is an inward flow of CSF along periarterial spaces;
The flow is driven across the layer of astrocyte endfeet into the parenchyma aided by the presence of Aqp4 in the endfeet;
The flow propels the waste products of metabolism into the perivenous space again crossing the layer of endfeet, presumably again aided by the presence of Aqp4;
The flow exits the parenchyma by the perivenous route and reaches lymphatic vessels in the neck.
As indicated when considering Proposal 1, a flow of ~ 0.6 µL g−1 min−1 or more would be required to remove the efflux markers at the observed rate. For a 1400 g brain, that is c. 1.2 L day−1 roughly twice the generally accepted rate of CSF production. Thus even if the rest of this proposal is correct, either the glymphatic flow does not direct ISF out of the brain directly to lymphatic vessels or the rate of CSF production is greater than is generally accepted.
The earlier evidence for and against the glymphatic hypothesis was discussed in [41] where it was argued that while a recirculation of CSF could explain influx and efflux of substances much faster than by simple diffusion, it did not explain either the observed outward movements of solutes along arteries [70, 71, 82, 83, 87, 130] or the observed continuation of rapid inward periarterial movement of large solutes when the proposed glymphatic circulation was interrupted at the level of the astrocyte endfeet by global knockout of Aqp4 [25].
Proposal 4 (not shown in Fig. 7) The most recent proposal [101] is that vasomotion, waxing and waning contraction of the smooth muscle fibres in the arterial wall, propels fluid towards the brain surface along the basement membranes of the vessel wall. This proposal does not seek to explain the rapid influx of markers along arterial walls, possibly by a different pathway.

Is movement within the parenchyma determined by diffusion or by flow from periarterial to perivenular spaces?
It is unclear how the flow required for the glymphatic hypothesis to be correct, at least 0.6 µL g−1 min−1 (see Proposal 3 above), could be driven through the parenchyma. Jin et al. [77] and Holter et al. [80] have calculated fluid flows within the parenchyma using, respectively, 2-D and 3-D models of the geometry and dimensions of the interstitial spaces. Jin et al. concluded that “little or no advective solute transport is predicted to occur with physiological paravascular pressure differences” taken to be < 5 mmHg. (Strictly advection corresponds to flow while convection includes both flow and diffusion). Furthermore they concluded that the water permeability of the endfeet membrane facing the microvessels, i.e. the membrane containing Aqp4, could have little direct effect on water flow into the parenchyma.7 Jin et al. assumed that the ISF between the cells behaves as a free fluid with the viscosity of water. If instead ISF in the interstitial spaces in the brain has properties similar to those of extracellular fluid in tissues in the rest of the body (see [138, 139], discussion in [41] and,8 the pressure required for flow would be much larger than that calculated by Jin et al. making bulk flow (advection) even less likely (compare [140]).
Holter et al. [80] have investigated what they consider to be a more realistic model of the parenchyma than that evaluated by Jin et al. One aspect is undeniably more realistic, it treats movement in three dimensions rather than two. It is also asserted that treating the obstacles to flow as being much smaller and more numerous than in Jin et al’s simulation produces a more faithful result. Jin et al. used barriers sized like cell bodies, while Holter et al. have adopted the smaller objects used in Kinney’s construction of the extracellular space [141], which allows for cell bodies and processes. (Smaller objects may be analogous to the increased resistance to flow resulting from macromolecules dissolved in peripheral extracellular fluid, see Footnote 8). Holter et al. conclude that flow makes a much smaller contribution than calculated by Jin et al. However, while Jin et al. treat the entrance and exit of fluid across the endfoot layers explicitly, this is missing from the treatment given by Holter et al. Given that the conclusion is “no flow” in both studies this difference between them may be of no consequence.
It should be noted that neither Jin et al. [77] nor Holter et al. [80] have considered flow along the basement membranes surrounding capillaries presumably because the total area available for such flow is less than for flow via the interstitial spaces (and flow along basement membranes wasn’t considered in the glymphatic hypothesis). Asgari et al. [73] assumed that the resistance to flow of the basement membranes would be the same as for slabs of ®Matrigel of the same dimensions, and on this basis concluded that flow via basement membranes would be less than through the interstitium (compare the discussion in [16]).
That flow through the parenchyma is not needed to explain the delivery of solutes to perivascular spaces was suggested by the results obtained using integrative optical imaging (see e.g. [24, 76, 142, 143]). That technique showed that in apparently isotropic regions of brain the spread of fluorescent indicators appears symmetrical over distances of at least 100 µm from a point source (for examples see [24]), indicating that molecules within ISF can reach perivascular spaces in any direction and in good time by diffusion with no evidence for preferential movement towards either arterioles or venules. However, that technique was applied using a water immersion microscope objective after opening the skull and dura to allow access [142]. The open skull and dura may have perturbed flow in the parenchyma. (There is good evidence that cisternal puncture changes flow in the basal cisterns and subarachnoid spaces [25, 89]). Symmetrical spread has now been convincingly confirmed in a systematic study using both direct observation through a cranial window after injection of fluorescently labelled dextrans and recovery from photobleaching [79]. However, it should be noted that the window was glazed after dye injection and hence only shortly before observations were made.
Smith et al. [79] have also found (1) that the dependence of the rate of movements within the parenchyma on the size of the solute is close to that expected if the movement occurs by diffusion; (2) that, in contrast to the report of Iliff et al. [25], the amounts of solutes entering the parenchyma are similar in Aqp4+/+ and Aqp4−/− mice; and (3) that local movement of solutes in the parenchyma is not impaired just after cardiorespiratory arrest. They conclude that “these results do not support glymphatic, convective solute transport in brain parenchyma.” In reply to point (2) a group of researchers have posted an un-refereed summary of their experience that comparing three different Aqp4 knockout transgenic lines, including the cell line used by Smith et al. [79], Aqp4 does support “fluid and solute transport and efflux in brain in accordance with the glymphatic system model” [144]. The role of Aqp4 is discussed further in [140].
Pizzo et al. [16] have looked at the distribution of IgG and much smaller single domain antibodies after cisternal infusion. They found that the antibodies rapidly enter the perivascular spaces of blood vessels of all sizes be they arteries, veins or capillaries. The distribution within the parenchyma was as expected for diffusion including the differences between the profiles for different sizes of fluorescent marker. Further discussion supporting the importance of diffusion over bulk flow in the extracellular spaces of the parenchyma can be found in [40]. Perivascular solute movements are considered further in Sect. 5.7.1.2.

Is there a glymphatic circulation?
The answer depends partly on what one means by glymphatic circulation. If the meaning is “Convective glymphatic fluxes of CSF and ISF propel the waste products of neuron metabolism into the paravenous space” [136], then the answer is almost certainly no (compare [40, 140], though it should be noted that [54, 137] still argue in favour of the original glymphatic hypothesis). However, if glymphatic circulation is taken to mean only that there is a net inward periarterial flow, a net outward perivenous flow, and some connection between them, then the answer still isn’t known with any certainty. The results discussed above [24, 76, 79, 142, 143] provide powerful experimental support for the widely held view that a glymphatic circulation is not needed to explain solute movements over the short distances that are important in the parenchyma. Furthermore the calculations of Asgari et al. [73, 78], Jin et al. [77] and Holter et al. [80] (see also Footnote 8) suggest that flow through the interstitial spaces of grey matter or along the basement membranes of microvessels in the parenchyma is negligible. However, it is not yet clear that the available experimental results exclude the possibility that there is a net flow between the perivascular spaces of arterioles and venules that is large enough to complete a recirculation pathway inwards from CSF via periarterial routes and back to CSF via perivenous routes.9 If that flow exists it could be important for transport of solutes over the relatively large distances encountered along the perivascular spaces (see e.g. [76]) while still being negligible relative to diffusion for transport over the relatively short distances within the parenchyma. Interestingly this scenario was proposed recently by Coles et al. [1] (see also Iliff et al. [145]) based on detailed consideration of the evidence available even before publication of the results in [16, 79].
While there have now been hundreds of references to the glymphatic mechanism, almost all of these treat it as accepted dogma and do not test the assumptions or the evidence on which it is based. At present it would be better to refer to perivascular elimination and delivery of substances without prejudice to the mechanism(s) by which these are achieved.

Variation between sleep and wakefulness
In the comparative studies undertaken on sleeping and awake mice by Xie et al. [128] there were differences in clearance and in interstitial fluid volume in the two physiological states. In these studies, inulin was used as the marker solute for perivascular clearance and the real-time iontophoresis method [74] was used to assess the volume. Briefly Xie et al. [128] found that, in the change from sleep to wakefulness, ISF volume decreased by 1.6-fold, the rate constant for efflux of inulin decreased 2.7-fold and from these values it could be estimated that inulin clearance decreased 4.3-fold (see Section 2.4 in [146]). Changes in the rate of access into the parenchyma of markers added to CSF and the discrepancies between the results of Xie et al. and of Gakuba et al. [147] are discussed briefly in.10
As discussed in [146] it is at present unclear whether any change in perivascular clearance of inulin in the transition from sleep to wakefulness is a consequence of the change in ISF volume in the parenchyma or some other effect. There are other possible effects of sleep versus wakefulness that might plausibly alter the clearance, e.g. changes in the shape or volume of either the perivascular spaces or the glial endfeet surrounding them.